Reagents and methods for detecting HCV

ABSTRACT

The present disclosure relates to oligonucleotide sequences for amplification primers and their use in performing nucleic acid amplifications of HCV, in particular regions that encode the NS3 polypeptide. In some embodiments the primers are used in nested PCR methods for the detection or sequencing of HCV NS3. The oligonucleotide sequences are also provided assembled as kits that can be used to amplify and detect or sequence HCV NS3.

PRIORITY CLAIM

This application claims priority to U.S. Provisional Patent ApplicationSer. Nos. 61/498,870, filed Jun. 20, 2011 and 61/645,149, filed May 10,2012; the entirety of both of which are hereby incorporated byreference.

This application incorporates by reference the sequence listing which issubmitted together with this application in computer readable form whichhas the file name SequenceListing_2011P13250WOUS.txt and is 10 KB.

BACKGROUND

Hepatitis C virus (HCV) infection is a major health problem that leadsto chronic liver disease, such as cirrhosis and hepatocellularcarcinoma, in a substantial number of infected individuals, estimated tobe 2-15% of the world's population. There are an estimated 3.9 millioninfected people in the United States alone, according to the U.S. Centerfor Disease Control, roughly five times the number of people infectedwith the human immunodeficiency virus (HIV). According to the WorldHealth Organization, there are more than 170 million infectedindividuals worldwide, with at least 3 to 4 million people beinginfected each year. Once infected, about 20% of people clear the virus,but the rest harbor HCV for the rest of their lives. Ten to twentypercent of chronically infected individuals eventually developliver-destroying cirrhosis or cancer. The viral disease is transmittedparenterally by contaminated blood and blood products, contaminatedneedles, or sexually, and vertically from infected mothers or carriermothers to their off-spring.

SUMMARY

The present disclosure relates to oligonucleotide sequences foramplification primers and their use in performing nucleic acidamplifications of HCV, in particular regions that encode the NS3polypeptide. In some embodiments the primers are used in nested PCRmethods for the detection or sequencing of HCV NS3. The oligonucleotidesequences are also provided assembled as kits that can be used to detector sequence HCV NS3.

In some embodiments, isolated oligonucleotide amplification primerscomprise a nucleic acid sequence selected from the group consisting ofSEQ ID NOS: 1-20, complementary sequences thereof, active fragmentsthereof, and combinations thereof.

In some embodiments, collections of primers for amplifying a portion ofHCV NS3 1a genomic sequence are provided which comprise one or moreforward primers selected from the group consisting of SEQ ID NO: 1, SEQID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, active fragmentsthereof, and combinations thereof; and one or more reverse primersselected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO: 8, SEQ ID NO: 9, SEQ ID NO: 10, active fragments thereof, andcombinations thereof.

In some embodiments, collections of primers for amplifying a portion ofHCV NS3 1b genomic sequence are provided which comprise one or moreforward primers selected from the group consisting of SEQ ID NO: 11, SEQID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, active fragmentsthereof, and combinations thereof; and one or more reverse primersselected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, active fragments thereof, andcombinations thereof.

In some embodiments, kits for amplifying HCV NS3 by nested PCR areprovided that comprise one of the aforementioned collections of primersas outer primers in combination with a suitable set of inner primers.

In some embodiments, methods of amplifying HCV NS3 in a sample areprovided that comprise contacting a sample with one of theaforementioned collections of primers as outer primers and submittingthe resulting mixture to a first nucleic acid amplification reaction. Insome embodiments, these methods further comprise contacting a productfrom the first nucleic acid amplification reaction with a set of innerprimers and submitting the resulting mixture to a second nucleic acidamplification reaction. In some embodiments, these methods furthercomprise sequencing a product from the second nucleic acid amplificationreaction to detect an HCV NS3 polymorphism.

BRIEF DESCRIPTION OF THE DRAWING

FIGS. 1A-D presents agarose gel electrophoretic profiles of first stageReverse Transcriptase (RT) PCR amplification products of 9 HCV 1aclinical isolates (Samples S1-S9). Each reaction was run in triplicateat 1,000 and/or 5,000 copies/ml. Also included were negative controlslacking HCV and a 1 kb ladder.

FIGS. 2A-C presents agarose gel electrophoretic profiles of a secondstage nested PCR amplification products of 9 HCV 1a clinical isolates(Samples S1-S9). Each reaction was run in triplicate and the startingtemplate was the amplification product of a RT PCR reaction with 1,000and/or 5,000 copies of HCV/ml. Also included were negative controlslacking HCV and a 1 kb ladder.

FIGS. 3A-B presents agarose gel electrophoretic profiles of first stageRT PCR amplification products of 6 HCV 1b clinical isolates (SamplesS10-S15). Each reaction was run in triplicate at 10,000 and/or 5,000copies/ml. Also included were negative controls lacking HCV and a 1 kbladder.

FIG. 4 presents agarose gel electrophoretic profiles of second stagenested PCR amplification products of 6 HCV 1b clinical isolates (SamplesS10-S15). Each reaction was run in triplicate and the starting templatewas the amplification product of a RT PCR reaction with 5,000 copies ofHCV/ml. Also included were negative controls lacking HCV and a 1 kbladder.

DEFINITIONS

The term “active fragment”, as used herein in reference to anoligonucleotide (e.g., an oligonucleotide sequence provided herein),refers to any nucleic acid molecule which includes fewer nucleotidesthan the full length oligonucleotide, and retains at least onebiological property of the full length oligonucleotide. For example, insome embodiments, active fragments may retain the ability to act asprimers in an HCV amplification reaction. An active fragment of thepresent disclosure can be a nucleic acid molecule which is, for example,10, 15, 20, 25, 30 or more nucleotides in length and can be used as aprimer in an HCV amplification reaction.

The term “amplification” or “amplification reaction” is used herein torefer to any in vitro process for exponentially increasing the number ofcopies of a nucleotide sequence or sequences. Nucleic acid amplificationresults in the incorporation of nucleotides (ribonucleotides ordeoxyribonucleotides) into primers to form DNA or RNA molecules that arecomplementary to a template nucleic acid molecule. As used herein, oneamplification reaction may consist of many rounds of primer extension.For example, one PCR reaction may consist of several cycles ofdenaturation and extension ranging from, e.g., about 5 cycles to about1000 cycles, or more.

The term “amplification reaction reagents”, is used herein to refer toreagents used in nucleic acid amplification reactions and may include,but are not limited to, buffers, enzymes having reverse transcriptaseand/or polymerase activity or exonuclease activity, enzyme cofactorssuch as magnesium or manganese, salts, nicotinamide adenine dinuclease(NAD) and deoxynucleoside triphosphates (dNTPs), such as deoxyadenosinetriphosphate, deoxyguanosine triphosphate, deoxycytidine triphosphateand deoxythymidine triphosphate.

The term “gene”, as used herein, has its art understood meaning, andrefers to a part of the genome specifying a macromolecular product, beit DNA for incorporation into a host genome, a functional RNA moleculeor a protein, and may include regulatory sequences (e.g., promoters,enhancers, etc.) and/or intron sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequences.

The term “hybridization”, as used herein, refers to the formation ofcomplexes (also called duplexes or hybrids) between nucleotide sequenceswhich are sufficiently complementary to form complexes via Watson-Crickbase pairing or non-canonical base pairing. It will be appreciated thathybridizing sequences need not have perfect complementary to providestable hybrids. In many situations, stable hybrids will form where fewerthan about 10% of the bases are mismatches. Accordingly, as used herein,the term “complementary” refers to a nucleic acid molecule that forms astable duplex with its complement under assay conditions, generallywhere there is about 90% or greater homology (e.g., about 95% orgreater, about 98% or greater, or about 99% or greater homology). Thoseskilled in the art understand how to estimate and adjust the stringencyof hybridization conditions such that sequences that have at least adesired level of complementarity will stably hybridize, while thosehaving lower complementarity will not. For examples of hybridizationconditions and parameters, see, for example, Sambrook et al., “MolecularCloning: A Laboratory Manual”, 1989, Second Edition, Cold Spring HarborPress: Plainview, N.Y. and Ausubel, “Current Protocols in MolecularBiology”, 1994, John Wiley & Sons: Secaucus, N.J. Complementaritybetween two nucleic acid molecules is said to be “complete”, “total” or“perfect” if all the nucleic acid's bases are matched, and is said to be“partial” otherwise.

The terms “labeled” and “labeled with a detectable agent (or moiety)”are used herein interchangeably to specify that an entity (e.g., atarget sequence) can be visualized, e.g., directly or followinghybridization to another entity that comprises a detectable agent ormoiety. Preferably, the detectable agent or moiety is selected such thatit generates a signal which can be measured and whose intensity isrelated to (e.g., proportional to) the amount of the entity of interest(e.g., a target sequence). Methods for labeling nucleic acid moleculesare well-known in the art. In some embodiments, labeled nucleic acidscan be prepared by incorporation of, or conjugation to, a label that isdirectly or indirectly detectable by spectroscopic, photochemical,biochemical, immunochemical, electrical, optical, or chemical means.

The term “melting temperature” or “Tm” of a specific oligonucleotide, asused herein, refers to the specific temperature at which half of theoligonucleotide hybridizes to its target in equilibrium. Accurateprediction of the Tm of any oligonucleotide can be made based onsequence using nearest neighbor parameter calculations.

The terms “nucleic acid”, “nucleic acid molecule”, “polynucleotide” or“oligonucleotide” are used herein interchangeably. They refer to linearpolymers of nucleotide monomers or analogs thereof, such asdeoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Unless otherwisestated, the terms encompass nucleic acid-like structures with syntheticbackbones, as well as amplification products. As will be appreciated byone skilled in the art, the length of these polymers (i.e., the numberof nucleotides it contains) can vary widely, often depending on theirintended function or use. In some embodiments, the term“oligonucleotide” is used herein to denote a polynucleotide thatcomprises between about 5 and about 150 nucleotides, e.g., between about10 and about 100 nucleotides, between about 15 and about 75 nucleotides,or between about 15 and about 50 nucleotides. Throughout thespecification, whenever an oligonucleotide is represented by a sequenceof letters (chosen, for example, from the four base letters: A, C, G,and T, which denote adenosine, cytidine, guanosine, and thymidine,respectively), the nucleotides are presented in the 5′→3′ order from theleft to the right. In some embodiments, the sequence of anoligonucleotide of the present disclosure contains the letter Y and/orletter R and/or letter N and/or letter W and/or letter B. As usedherein, the letter “Y” represents a degenerative base, which can be T orC with substantially equal probability. As used herein, the letter “R”represents a degenerative base, which can be A or G with substantiallyequal probability. As used herein, the letter “N” represents adegenerative base, which can be A or G or T or C. As used herein, theletter “W” represents a degenerative base, which can be A or T. As usedherein, the letter “B” represents a degenerative base, which can be C orG or T. Thus, for example, in the context of the present disclosure, ifan oligonucleotide contains one degenerative base M, the oligonucleotideis a substantially equimolar mixture of two subpopulations of a firstoligonucleotide where the degenerative base is A and a secondoligonucleotide where the degenerative base is C, the first and secondoligonucleotides being otherwise identical.

The term “3′” refers to a region or position in a polynucleotide oroligonucleotide 3′ (i.e., downstream) from another region or position inthe same polynucleotide or oligonucleotide. The term “5′” refers to aregion or position in a polynucleotide or oligonucleotide 5′ (i.e.,upstream) from another region or position in the same polynucleotide oroligonucleotide. The terms “3′ end” and “3′ terminus”, as used herein inreference to a nucleic acid molecule, refer to the end of the nucleicacid which contains a free hydroxyl group attached to the 3′ carbon ofthe terminal pentose sugar. The term “5′ end” and “5′ terminus”, as usedherein in reference to a nucleic acid molecule, refers to the end of thenucleic acid molecule which contains a free hydroxyl or phosphate groupattached to the 5′ carbon of the terminal pentose sugar.

The term “isolated”, as used herein in reference to an oligonucleotide,means an oligonucleotide, which by virtue of its origin or manipulation,is separated from at least some of the components with which it isnaturally associated or with which it is associated when initiallyobtained. By “isolated”, it is alternatively or additionally meant thatthe oligonucleotide of interest is produced or synthesized by the handof man.

The terms “primer”, as used herein, typically refers to oligonucleotidesthat hybridize in a sequence specific manner to a complementary nucleicacid molecule (e.g., a nucleic acid molecule comprising a targetsequence). In some embodiments, a primer will comprise a region ofnucleotide sequence that hybridizes to at least about 8, e.g., at leastabout 10, at least about 15, or about 20 to about 40 consecutivenucleotides of a target nucleic acid (i.e., will hybridize to acontiguous sequence of the target nucleic acid). In general, a primersequence is identified as being either “complementary” (i.e.,complementary to the coding or sense strand (+)), or “reversecomplementary” (i.e., complementary to the anti-sense strand (−)). Insome embodiments, the term “primer” may refer to an oligonucleotide thatacts as a point of initiation of a template-directed synthesis usingmethods such as PCR (polymerase chain reaction) or LCR (ligase chainreaction) under appropriate conditions (e.g., in the presence of fourdifferent nucleotide triphosphates and a polymerization agent, such asDNA polymerase, RNA polymerase or reverse-transcriptase, DNA ligase,etc., in an appropriate buffer solution containing any necessaryreagents and at suitable temperature(s)). Such a template directedsynthesis is also called “primer extension”. For example, a primer pairmay be designed to amplify a region of DNA using PCR. Such a pair willinclude a “forward primer” and a “reverse primer” that hybridize tocomplementary strands of a DNA molecule and that delimit a region to besynthesized and/or amplified.

The terms “forward primer” and “forward amplification primer” are usedherein interchangeably, and refer to a primer that hybridizes (oranneals) to the target (template) strand. The terms “reverse primer” and“reverse amplification primer” are used herein interchangeably, andrefer to a primer that hybridizes (or anneals) to the complementarytarget strand. The forward primer hybridizes with the target sequence 5′with respect to the reverse primer.

The term “primer set” is used herein to refer to two or more primerswhich together are capable of priming the amplification of a targetnucleotide sequence (e.g., to amplify DNA or RNA encoding HCV NS3 or aportion thereof). In some embodiments, the term “primer set” refers to apair of primers including a 5′ (upstream) primer (or forward primer)that hybridizes with the 5′-end of the nucleic acid sequence to beamplified and a 3′ (downstream) primer (or reverse primer) thathybridizes with the complement of the sequence to be amplified. Suchprimer set or primer pair are particularly useful in PCR amplificationreactions.

As used herein, the term “nested primer set” refers to two or moreprimers which together are capable of priming the amplification of anamplified nucleotide sequence of interest. The primers in a “nestedprimer set” are sometimes referred to herein as “inner primers”. In someembodiments, one or more primers of the “nested primer set” areoverlapping with primers that were used to amplify the originalnucleotide sequence of interest (i.e., with “outer primers”). In someembodiments, the “nested primer set” is non-overlapping with primersthat were used to amplify the original nucleotide sequence of interest.In some embodiments, the term “nested primer set” refers to a pair ofprimers including a 5′ (upstream) primer (or forward primer) thathybridizes with or towards the 5′-end of the amplified nucleic acidsequence of interest and a 3′ (downstream) primer (or reverse primer)that hybridizes with or towards the 5′-end of the complement of theamplified nucleic acid sequence of interest.

As used herein, the term “sample” refers to a biological sample obtainedor derived from a source of interest, as described herein. In someembodiments, a source of interest comprises an organism, such as ananimal or human. In some embodiments, a biological sample comprisesbiological tissue or fluid. In some embodiments, a biological sample maybe or comprise bone marrow; blood; blood cells; ascites; tissue or fineneedle biopsy samples; cell-containing body fluids; free floatingnucleic acids; sputum; saliva; urine; cerebrospinal fluid, peritonealfluid; pleural fluid; feces; lymph; gynecological fluids; skin swabs;vaginal swabs; oral swabs; nasal swabs; washings or lavages such as aductal lavages or broncheoalveolar lavages; aspirates; scrapings; bonemarrow specimens; tissue biopsy specimens; surgical specimens; feces,other body fluids, secretions, and/or excretions; and/or cellstherefrom, etc. In some embodiments, a biological sample is or comprisescells obtained from an individual. In some embodiments, obtained cellsare or include cells from an individual from whom the sample isobtained. In some embodiments, obtained cells are or include microbialcells of an individual's microbiome. In some embodiments, a sample is a“primary sample” obtained directly from a source of interest by anyappropriate means. For example, in some embodiments, a primarybiological sample is obtained by methods selected from the groupconsisting of biopsy (e.g., fine needle aspiration or tissue biopsy),surgery, collection of body fluid (e.g., blood, lymph, feces etc.), etc.In some embodiments, as will be clear from context, the term “sample”refers to a preparation that is obtained by processing (e.g., byremoving one or more components of and/or by adding one or more agentsto) a primary sample. For example, filtering using a semi-permeablemembrane. Such a “secondary sample” or “processed sample” may comprise,for example nucleic acids or proteins extracted from a “primary sample”or obtained by subjecting a “primary sample” to techniques such asamplification or reverse transcription of mRNA, isolation and/orpurification of certain components, etc.

The term “target nucleic acid sequence” or “nucleic acid of interest” isused herein to refer to any series of contiguous nucleotides in atemplate nucleic acid molecule (such as DNA, cDNA or RNA) to beamplified. One specific target nucleic acid sequence is a segment,region, or fragment of a nucleic acid molecule that hybridizes to atleast one inner primer during a nested PCR reaction.

DETAILED DESCRIPTION OF SOME EMBODIMENTS

HCV is an enveloped, positive stranded RNA virus which has beenclassified as a separate genus within the Flavivirus family, e.g., seeHeinz, Arch. Virol. (Suppl.) 4:163 (1992). The viral genome isapproximately 9,500 nucleotides in length and contains one long openreading frame that encodes a precursor polyprotein of 330 Kd. IndividualHCV polypeptides are produced by proteolytic processing of the precursorpolypeptide. This proteolysis is catalyzed by a combination of bothcellular and viral encoded proteases.

The organization and processing of the HCV polyprotein appears to bemost similar to that of the pestiviruses. At least 10 polypeptides havebeen identified and the order of these cleavage products in thepolyprotein is NH₂—C-E1-E2-p7-NS2-NS3-NS4A-NS4B—NS5A-NS5B—COOH.Proteolytic processing is mediated by host signal peptidase and twoHCV-encoded proteinases, the NS2-3 autoproteinase and the NS3-4A serineproteinase. For further details see Rice, in “Fields Virology” (Fieldset al., Eds.), pp. 931-960, Raven Press, New York (1996) and Shimotohnoet al., J. Hepatol. 22: 87-92 (1995).

C is a basic protein that serves as the viral core or capsid protein; E1and E2 are virion envelope glycoproteins; p7 is a hydrophobic protein ofunknown function that is inefficiently cleaved from the E2 glycoprotein,e.g., see Mizushima et al., J. Virol. 68: 6215-6222 (1994) and Selby etal., Virology 204: 114-122 (1994). NS2 to NS5B are nonstructural (NS)proteins which function in viral RNA replication complexes. Theirfunctions have been identified as follows: NS2 is a metalloprotease; NS3is a protease/helicase that contains motifs characteristic of RNAhelicases and that has been shown to possess an RNA-stimulated NTPaseactivity, e.g., see Suzich et al., J. Virol. 67, 6152-6158 (1993); NS4Ais a co-factor for NS3; NS4B is of unknown function; NS5A interacts withcellular factors to transcriptionally modulate cellular genes andpromote cell growth, e.g., see Ghosh et al., J. Biol. Chem.275:7184-7188 (2000) and provide IFNα resistance; and NS5B is areplicase that contains the GDD motif characteristic of theRNA-dependent RNA polymerases of other positive-strand RNA viruses.

Several HCV polypeptides are putative targets for therapeuticintervention, including a metalloprotease (NS2-3), a serine protease(NS3), a helicase (NS3), and an RNA-dependent RNA polymerase (NS5B). TheNS3 protease is located in the N-terminal domain of the NS3 protein, andis considered a prime drug target since it is responsible for anintramolecular cleavage at the NS3/4A site and for downstreamintermolecular processing at the NS4A/4B, NS4B/5A and NS5A/5B junctions.

I—Oligonucleotide Sequences and Amplification Primer Sets

Oligonucleotide Sequences

Previous research has identified subtypes of HCV with varyingsusceptibility to treatment. For example, pegylated-interferon incombination with Ribavirin, exhibits a sustained response rate of 40 to50% across HCV patients. However, a majority of patients still do notelicit a sustained anti-viral response, particularly against theinterferon-resistant HCV genotypes, 1a and 1b. The present disclosureprovides oligonucleotide sequences that are specific for the 1a and 1bgenotypes of the HCV NS3 gene.

Existing DNA sequencing assays for HCV NS3 region have limitedperformance due to the high variability of the HCV genome. The main modeof failure is due to poor performance of oligonucleotides meant toinitiate reverse transcription followed by PCR based amplification ofthe viral RNA. There is a need to improve the sensitivity performancebeyond what is currently possible.

Previous assays have not demonstrated sufficient performance to bewidely accepted in clinical laboratories and have not been submitted forregulatory approval. Most are used as research assays or local home-brewlaboratory developed tests due to these performance limitations.Previous assay designs have not solved these limitations.

The present disclosure, in contrast, results in uniquely high performingassay design prototypes. The top performing design meets IVDcommercialization criteria of >95% successful amplification with knownglobal sequence variants. This performance criteria has been previouslydemonstrated to be indicative of sufficient sensitivity across geneticvariants to be implemented for routine clinical use with samples ininternational studies.

For the particular region targeted, HCV NS3, the design that wasdeveloped is likely to exhibit unique performance compared to otherpossible designs for the purpose of in vitro reverse transcription andPCR amplification of the entire NS3 coding region for genotype 1,subtypes 1a and 1b of HCV. This amplicon is considered the idealmaterial for further analysis by direct DNA sequencing, or nested PCRfollowed by DNA sequencing, to determine mutations associated withresistance to protease inhibitors in the NS3 region of HCV. It is alsoideal for cloning and expression in model organisms for HCV diseasestudies and pharmaceutical development programs. The amplifiedpatient-derived product may also be cloned for in vitro phenotypingexperiments with pseudotyped virus.

The sequences of certain oligonucleotides of the present disclosure areset forth in Table 1 (SEQ ID NOS: 1-45).

TABLE 1 SEQ ID Sequence NO. Name Sequence (5′ → 3′) Strand 1 NS3_1a_1FAGGGTGGAGGTTGCTGGC (+) 2 NS3_1a_2F GGGGTGGAGATTGCTGGC (+) 3 NS3_1a_3FGGGATGGAGGTTGCTGGC (+) 4 NS3_1a_4F GGGGTGGAGGTTGTTGGC (+) 5 NS3_1a_5FGGGGTGGAGGTTGCTGGC (+) 6 NS3_1a_6R GCCGGAACCTTGGTGCTCT (−) 7 NS3_1a_7RGCCGGGACTTTGGTGCTTT (−) 8 NS3_1a_8R GCTGGGACCTTGGTGCTCT (−) 9 NS3_1a_9RGCCGGGACCTTGGTGCTCT (−) 10 NS3_1a_10R GCCGGGACCTTGGTGCTTT (−) 11NS3_1b_1F AGCCTCACAGGCCGGGAC (+) 12 NS3_1b_2F AGCCTTACAGGCCGGGAC (+) 13NS3_1b_3F AGTCTCACAGGCCGGGAC (+) 14 NS3_1b_4F AGTCTCACAGGGCGGGAC (+) 15NS3_1b_5F AGCCTCACAGGTCGGGAT (+) 16 NS3_1b_6R GCCACTTGGAATGTTTGCGG (−)17 NS3_1b_7R GCCACTTGGAATGTCTGCGG (−) 18 NS3_1b_8R GCCACCTGGAATGTCTGCGG(−) 19 NS3_1b_9R GCCACTTGGAATGACTGCGG (−) 20 NS3_1b_10RGCCACTTGGAAAGTCTGCGG (−) 21 SCR6.1 GCTTGGGATATGATGATGAACTGGTC (+) 22SCR6.2 GCGTGGGATATGATGATGAACTGGTC (+) 23 SCR6.3GCATGGGATATGATGATGAACTGGTC (+) 24 SCR6.4 GCCTGGGATATGATGATGAACTGGTC (+)25 SCR8 TGGGATATGATGATGAACTGGTC (+) 26 SCR4.1 AGGAACTTGCCGTAGGTGGAGTA(−) 27 SCR4.2 AGGAACTTGCCGTAGGTGGAATA (−) 28 SCR4.3AGAAACTTGCCGTAGGTGGAGTA (−) 29 SCR4.4 AGAAACTTGCCGTAGGTGGAGTA (−) 30SCR7.1 AGGAACTTGCCGTAGGTGGA (−) 31 SCR7.2 AGGAACTTGCCATAGGTGGA (−) 32SCR7.3 AGAAACTTGCCGTAGGTGGA (−) 33 NS3-899s GACTGGCCGGGACAARAACC (+) 34NS3-898as GCTGGTGGRGAGGARTTGTC (−) 35 NS3-892s GTATCATCACYAGCCTCACRG (+)36 NS3-893as CACTTGGAATGTYTGCGGTAC (−) 37 BP_M13_FwdTTCTGGCGTACCGTTCCTGT (+) 38 BP_M13_Rev GTTTTCCCAGTCACGACGTTGTA (−) 39NS3_1a_F RGGRTGGAGRTTGYTGGC (+) 40 NS3_1a_R GCYGGRACYTTGGTGCTYT (−) 41NS3_1b_F AGYCTYACAGGBCGGGAY (+) 42 NS3_1b_R GCCACYTGGAAWGWYTGCGG (−) 43SCR6 GCNTGGGATATGATGATGAACTGGTC (+) 44 SCR4 AGRAACTTGCCRTAGGTGGARTA (−)45 SCR7 AGRAACTTGCCRTAGGTGGA (−)

In some embodiments, primers are degenerate primers encompassing thesequences of other primers. SEQ ID NO 39 comprises a degenerate primersequence encompassing the sequences of SEQ ID NOS: 1-5. SEQ ID NO 40comprises a degenerate primer sequence encompassing the sequences of SEQID NOS: 6-10. SEQ ID NO 41 comprises a degenerate primer sequenceencompassing the sequences of SEQ ID NOS: 11-15. SEQ ID NO 42 comprisesa degenerate primer sequence encompassing the sequences of SEQ ID NOS:16-20. SEQ ID NO 43 comprises a degenerate primer sequence encompassingthe sequences of SEQ ID NOS: 21-25. SEQ ID NO 44 comprises a degenerateprimer sequence encompassing the sequences of SEQ ID NOS: 26-29. SEQ IDNO 45 comprises a degenerate primer sequence encompassing the sequencesof SEQ ID NOS: 30-32.

Amplification Primer Sets

Oligonucleotides of the present disclosure may be conveniently providedas primer sets that can be used to amplify an HCV NS3 gene, e.g., todetermine which polymorphic variant(s) is/are present among some or allof the possible polymorphic variants that may exist at a particularpolymorphic site. Multiple sets of primers, capable of detectingpolymorphic variants at a plurality of polymorphic sites are provided.

Examples of primer sets/pairs comprising a forward amplification primerand a reverse amplification primer include:

-   -   Primer Set 1, which comprises forward primers comprising all of        SEQ ID NOS: 1-5 or any active fragments thereof and reverse        primers comprising all of SEQ ID NOS: 6-10 or any active        fragments thereof.    -   Primer Set 2, which comprises forward primers comprising all of        SEQ ID NOS: 11-15 or any active fragments thereof and reverse        primers comprising all of SEQ ID NOS: 16-20 or any active        fragments thereof.    -   Primer Set 3, which comprises a forward primer comprising SEQ ID        NO: 21 or any active fragment thereof and a reverse primer        comprising SEQ ID NO: 26 or any active fragment thereof.    -   Primer Set 4, which comprises forward primers comprising all of        SEQ ID NOS: 21-25 or any active fragments thereof and reverse        primers comprising all of SEQ ID NOS: 26-29 or any active        fragments thereof.    -   Primer Set 5, which comprises a forward primer comprising SEQ ID        NO: 21 or any active fragment thereof and reverse primers        comprising all of SEQ ID NOS: 26-29 or any active fragments        thereof.    -   Primer Set 6, which comprises forward primers comprising all of        SEQ ID NOS: 21-25 or any active fragments thereof and a reverse        primer comprising SEQ ID NO: 26 or any active fragment thereof.    -   Primer Set 7, which comprises a forward primer comprising SEQ ID        NO: 25 or any active fragment thereof and a reverse primer        comprising SEQ ID NO: 30 or any active fragment thereof.    -   Primer Set 8, which comprises a forward primer comprising SEQ ID        NO: 25 or any active fragment thereof and reverse primers        comprising all of SEQ ID NOS: 30-32 or any active fragments        thereof.    -   Primer Set 9, which comprises a forward primer comprising SEQ ID        NO: 33 or any active fragment thereof and a reverse primer        comprising SEQ ID NO: 34 or any active fragment thereof.    -   Primer Set 10, which comprises a forward primer comprising SEQ        ID NO: 35 or any active fragment thereof and a reverse primer        comprising SEQ ID NO: 36 or any active fragment thereof.    -   Primer Set 11, which comprises a forward primer comprising SEQ        ID NO: 39 or any active fragment thereof and a reverse primer        comprising SEQ ID NO: 40 or any active fragment thereof.    -   Primer Set 12, which comprises a forward primer comprising SEQ        ID NO: 41 or any active fragment thereof and a reverse primer        comprising SEQ ID NO: 42 or any active fragment thereof.    -   Primer Set 13, which comprises a forward primer comprising SEQ        ID NO: 43 or any active fragment thereof and a reverse primer        comprising SEQ ID NO: 44 or any active fragment thereof.    -   Primer Set 14, which comprises a forward primer comprising SEQ        ID NO: 25 or any active fragment thereof and a reverse primer        comprising SEQ ID NO: 45 or any active fragment thereof.        Oligonucleotide Preparation

Oligonucleotides of the present disclosure may be prepared by any of avariety of methods (see, e.g., Sambrook et al., “Molecular Cloning: ALaboratory Manual”, 1989, 2^(nd) Ed., Cold Spring Harbour LaboratoryPress: New York, N.Y.; “PCR Protocols: A Guide to Methods andApplications”, 1990, Innis (Ed.), Academic Press: New York, N.Y.;Tijssen “Hybridization with Nucleic Acid Probes—Laboratory Techniques inBiochemistry and Molecular Biology (Parts I and II)”, 1993, ElsevierScience; “PCR Strategies”, 1995, Innis (Ed.), Academic Press: New York,N.Y.; and “Short Protocols in Molecular Biology”, 2002, Ausubel (Ed.),5^(th) Ed., John Wiley & Sons: Secaucus, N.J.).

In some embodiments, oligonucleotides may be prepared by chemicaltechniques well-known in the art, including, e.g., chemical synthesisand polymerization based on a template as described, e.g., in Narang etal., Meth. Enzymol. 68:90-98 (1979); Brown et al., Meth. Enzymol. 68:109-151 (1979); Belousov et al., Nucleic Acids Res. 25:3440-3444 (1997);Guschin et al., Anal. Biochem. 250:203-211 (1997); Blommers et al.,Biochemistry 33:7886-7896 (1994); Frenkel et al., Free Radic. Biol. Med.19:373-380 (1995); and U.S. Pat. No. 4,458,066.

In some embodiments, oligonucleotides may be prepared using anautomated, solid-phase procedure based on the phosphoramidite approach.In such methods, each nucleotide is individually added to the 5′-end ofthe growing oligonucleotide chain, which is attached at the 3′-end to asolid support. The added nucleotides are in the form of trivalent3′-phosphoramidites that are protected from polymerization by adimethoxytriyl (or DMT) group at the 5′-position. After base-inducedphosphoramidite coupling, mild oxidation to give a pentavalentphosphotriester intermediate and DMT removal provides a new site foroligonucleotide elongation. The oligonucleotides are then cleaved offthe solid support, and the phosphodiester and exocyclic amino groups aredeprotected with ammonium hydroxide. These syntheses may be performed onoligo synthesizers such as those commercially available from PerkinElmer/Applied Biosystems, Inc. (Foster City, Calif.), DuPont(Wilmington, Del.) or Milligen (Bedford, Mass.). Alternatively,oligonucleotides can be custom made and ordered from a variety ofcommercial sources well-known in the art, including, for example, theMidland Certified Reagent Company (Midland, Tex.), ExpressGen, Inc.(Chicago, Ill.), Operon Technologies, Inc. (Huntsville, Ala.), and manyothers.

Purification of oligonucleotides, where necessary or desirable, may becarried out by any of a variety of methods well-known in the art. Forexample, purification of oligonucleotides is typically performed eitherby native acrylamide gel electrophoresis, by anion-exchange HPLC, e.g.,see Pearson and Regnier, J. Chrom. 255:137-149 (1983) or by reversephase HPLC, e.g., see McFarland and Borer, Nucleic Acids Res.7:1067-1080 (1979).

The sequence of oligonucleotides can be verified using any suitablesequencing method including, but not limited to, chemical degradation,e.g., see Maxam and Gilbert, Methods of Enzymology, 65:499-560 (1980),matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)mass spectrometry, e.g., see Pieles et al., Nucleic Acids Res.21:3191-3196 (1993), mass spectrometry following a combination ofalkaline phosphatase and exonuclease digestions, e.g., see Wu andAboleneen, Anal. Biochem. 290:347-352 (2001).

The present disclosure encompasses modified versions of theseoligonucleotides that perform as equivalents of these oligonucleotidesin accordance with the methods of the present disclosure. These modifiedoligonucleotides may be prepared using any of several means known in theart. Non-limiting examples of such modifications include methylation,“caps”, substitution of one or more of the naturally occurringnucleotides with an analog, and internucleotide modifications such as,for example, those with uncharged linkages (e.g., methyl phosphonates,phosphotriesters, phosphoroamidates, carbamates, etc.), or chargedlinkages (e.g., phosphorothioates, phosphorodithioates, etc.). Modifiedoligonucleotide may also be derivatized by formation of a methyl orethyl phosphotriester or an alkyl phosphoramidate linkage. Furthermore,the oligonucleotides of the present disclosure may also be modified witha label.

Labeling of Oligonucleotides

In some embodiments, the primers are labeled with a detectable agent ormoiety before being used in amplification/detection assays. The role ofa detectable agent is to allow visualization and detection of amplifiedtarget sequences. Preferably, the detectable agent is selected such thatit generates a signal which can be measured and whose intensity isrelated (e.g., proportional) to the amount of amplification products inthe sample being analyzed.

The association between the oligonucleotide and the detectable agent canbe covalent or non-covalent. Labeled detection primers can be preparedby incorporation of or conjugation to a detectable moiety. Labels can beattached directly to the nucleic acid sequence or indirectly (e.g.,through a linker). Linkers or spacer arms of various lengths are knownin the art and are commercially available, and can be selected to reducesteric hindrance, or to confer other useful or desired properties to theresulting labeled molecules, e.g., see Mansfield et al., Mol. CellProbes 9:145-156 (1995).

Various methods for labeling nucleic acid molecules are known in theart. For a review of labeling protocols, label detection techniques, andrecent developments in the field, see, for example, Kricka, Ann. Clin.Biochem. 39:114-129 (2002); van Gijlswijk et al., Expert Rev. Mol.Diagn. 1:81-91 (2001); and Joos et al., J. Biotechnol. 35:135-153(1994). Standard nucleic acid labeling methods include: incorporation ofradioactive agents, direct attachments of fluorescent dyes (Smith etal., Nucl. Acids Res. 13:2399-2412 (1985)) or of enzymes (Connoly andRider, Nucl. Acids. Res. 13:4485-4502 (1985)); chemical modifications ofnucleic acid molecules making them detectable immunochemically or byother affinity reactions, e.g., see Broker et al., Nucl. Acids Res.5:363-384 (1978); Bayer et al., Methods of Biochem. Analysis 26:1-45(1980); Langer et al., Proc. Natl. Acad. Sci. USA 78:6633-6637 (1981);Richardson et al., Nucl. Acids Res. 11:6167-6184 (1983); Brigati et al.,Virol. 126:32-50 (1983); Tchen et al., Proc. Natl. Acad. Sci. USA81:3466-3470 (1984); Landegent et al., Exp. Cell Res. 15:61-72 (1984);and Hopman et al., Exp. Cell Res. 169:357-368 (1987); andenzyme-mediated labeling methods, such as random priming, nicktranslation, PCR and tailing with terminal transferase. For a review onenzymatic labeling, see, e.g., Temsamani and Agrawal, Mol. Biotechnol.5:223-232 (1996). More recently developed nucleic acid labeling systemsinclude, but are not limited to: ULS (Universal Linkage System), whichis based on the reaction of monoreactive cisplatin derivatives with theN7 position of guanine moieties in DNA (Heetebrij et al., Cytogenet.Cell. Genet. 87:47-52 (1999)), psoralen-biotin, which intercalates intonucleic acids and upon UV irradiation becomes covalently bonded to thenucleotide bases (Levenson et al., Methods Enzymol. 184:577-583 (1990);and Pfannschmidt et al., Nucleic Acids Res. 24:1702-1709 (1996)),photoreactive azido derivatives (Neves et al., Bioconjugate Chem.11:51-55 (2000)), and DNA alkylating agents (Sebestyen et al., Nat.Biotechnol. 16: 568-576 (1998)).

It will be appreciated that any of a wide variety of detectable agentscan be used in the practice of the present disclosure. Suitabledetectable agents include, but are not limited to, various ligands,radionuclides (such as for example, ³²P, ³⁵S, ³H, ¹⁴C, ¹²⁵I, ¹³¹I, andthe like); fluorescent dyes; chemiluminescent agents (such as, forexample, acridinium esters, stabilized dioxetanes, and the like);spectrally resolvable inorganic fluorescent semiconductor nanocrystals(i.e., quantum dots), metal nanoparticles (e.g., gold, silver, copperand platinum) or nanoclusters; enzymes (such as, for example, those usedin an ELISA, e.g., horseradish peroxidase, beta-galactosidase,luciferase, alkaline phosphatase); colorimetric labels (such as, forexample, dyes, colloidal gold, and the like); magnetic labels (such as,for example, Dynabeads™); and biotin, dioxigenin or other haptens andproteins for which antisera or monoclonal antibodies are available.

A “tail” of normal or modified nucleotides can also be added to tag anoligonucleotide for detectability purposes. In some embodiments, an M13tag sequence (SEQ ID NO: 37 or 38) may be added.

II—Amplification Methods

In some embodiments, the present disclosure provides methods that usethe aforementioned oligonucleotides as amplification primers to amplifyregions of the HCV genome, in particular regions that encode the NS3polypeptide. As discussed in more detail below, in some embodiments theprimers are used in nested PCR methods for the amplification anddetection or sequencing of HCV NS3.

Preparation of RNA

In some embodiments, the disclosed methods may involve some level of RNApreparation. Indeed, the template for an amplification reaction (e.g., aPCR reaction) is typically DNA and the target HCV material to beanalyzed is typically expressed viral RNA. As a result, the startingtemplate material for the amplification reaction will often be cDNAwhich was generated from purified RNA including RNA from viruses. TheRNA preparation step may be performed far removed from the actualamplification step, for example, in another laboratory, or at a muchearlier time; however, in some embodiments the RNA isolation andpreparation of the cDNA may occur in conjunction with the amplificationstep of the methods.

When an RNA preparation step is included in the disclosed methods, themethod of RNA preparation can be any method of RNA preparation thatproduces enzymatically manipulatable mRNA. For example, the RNA can beisolated by using the guanidinium isothiocyanate-ultracentrifugationmethod, the guanidinium and phenol-chloroform method, the lithiumchloride-SDS-urea method or poly A+/mRNA from tissue lysates usingoligo(dT) cellulose method, e.g., see Schildkraut et al., J. Mol. Biol.4, 430-433 (1962); Chomczynski and Sacchi, Anal. Biochem. 162:156(1987); Auffray and Rougeon, Eur. J. Biochem. 107:303-314 (1980); Avivand Leder, Proc. Natl. Acad. Sci. USA 69, 1408-1412 (1972); and Sambrooket al., Selection of poly A+ RNA in “Molecular Cloning”, Vol. 1,7.26-7.29 (1989).

RNA can be isolated from any desired cell or cell type and from anyorganism, including mammals, such as mouse, rat, rabbit, dog, cat,monkey, and human, as well as other non-mammalian animals, such as fishor amphibians, as well as plants and even prokaryotes, such as bacteria.Thus, the DNA used in the method can also be from any organism, such asthat disclosed for RNA.

Generation of cDNA

In some embodiments, disclosed methods involve cDNA preparation. ThecDNA preparation step may be performed far removed from the actualamplification step, for example, in another laboratory, or at a muchearlier time; however, in some embodiments the preparation of the cDNAmay occur in conjunction with the amplification step of the methods.

When a cDNA preparation step is included in the disclosed methods, themethod of cDNA preparation can be any method of cDNA preparation thatproduces enzymatically manipulatable cDNA. For example, the cDNA can beprepared by using, for example, random primers, poly-d(T) oligos, orNVd(T) oligos. For the purpose of data normalization, an equal amount oftotal RNA is typically used for cDNA synthesis. Many examples exist ofperforming reverse transcription to produce cDNA for use in PCR,including the following: Glisin et al., Biochemistry 13:2633-7 (1974);Ullrich et al., Science 196:1313 (1977); Chirgwin et al., Biochemistry18:5294-9 (1979); Faulkner-Jones et al., Endocrinol. 133:2962-2972(1993); and Gonda et al., Mol. Cell Biol. 2:617-624 (1982).

Reverse transcriptases from any source (native or recombinant) may beused in the practice of the present disclosure. Suitable reversetranscriptases include, but are not limited to, those from Moloneymurine leukemia virus (M-MLV), human T-cell leukemia virus type I(HTLV-I), bovine leukemia virus (BLV), Avian Sarcoma Leukemia Viruses(ASLV) including Rous Sarcoma Virus (RSV) and Avian Myeloblastosis Virus(AMV), human immunodeficiency virus (HIV), cauliflower mosaic virus,Saccharomyces, Neurospora, Drosophila, primates, and rodents. See, forexample, U.S. Pat. Nos. 4,663,290 and 6,063,60; Grandgenett, et al.,Proc. Nat. Acad. Sci. (USA) 70:230-234 (1973), Gerard, DNA 5:271-279(1986), Kotewicz, et al., Gene 35:249-258 (1985), Tanese et al., Proc.Natl. Acad. Sci. (USA) 82:4944-4948 (1985), Roth et al., J. Biol. Chem.260:9326-9335 (1985), Michel et al., Nature 316:641-643 (1985), Akins etal., Cell 47:505-516 (1986) and EMBO J. 4:1267-75 (1985), and Fawcett,Cell 47:1007-1015 (1986); Shinnick et al., Nature 293:543-548 (1981);Seiki et al., Proc. Natl. Acad. Sci. USA 80:3618-3622 (1983); Rice etal., Virology 142:357-77 (1985); Schwartz et al., Cell 32:853-869(1983); Larder et al., EMBO J. 6:3133-3137 (1987); Farmerie et al.,Science 236:305-308 (1987); Barr et al., Biotechnology 5:486-489(1987)); Tanese et al., J. Virol. 59:743-745 (1986); Hansen et al., J.Biol. Chem. 262:12393-12396 (1987); Sonigo et al., Cell 45:375-85(1986); Takatsuji et al., Nature 319:240-243 (1986); Toh et al., Nature305:827-829 (1983)); Alexander et al., J. Virol. 61:534-542 (1987); andYuki et al., Nucl. Acids Res. 14:3017-3030 (1986).

Amplification Reaction

The use of oligonucleotide sequences of the present disclosure asprimers to amplify HCV NS3 target sequences in test samples is notlimited to any particular nucleic acid amplification technique or anyparticular modification thereof. In fact, the inventive oligonucleotidesequences can be employed in any of a variety of nucleic acidamplification methods well-known in the art (see, for example, Kimmeland Berger, Methods Enzymol. 152: 307-316 (1987); Sambrook et al.,“Molecular Cloning: A Laboratory Manual”, 1989, 2^(nd) Ed., Cold SpringHarbour Laboratory Press: New York, N.Y.; “Short Protocols in MolecularBiology”, Ausubel (Ed.), 2002, 5^(th) Ed., John Wiley & Sons: Secaucus,N.J.).

Such nucleic acid amplification methods include, but are not limited to,the Polymerase Chain Reaction (or PCR, described, for example, in “PCRProtocols: A Guide to Methods and Applications”, Innis (Ed.), 1990,Academic Press: New York; “PCR Strategies”, Innis (Ed.), 1995, AcademicPress: New York; “Polymerase chain reaction: basic principles andautomation in PCR: A Practical Approach”, McPherson et al. (Eds.), 1991,IRL Press: Oxford; Saiki et al., Nature 324:163 (1986); and U.S. Pat.Nos. 4,683,195, 4,683,202 and 4,889,818, each of which is incorporatedherein by reference in its entirety); and reverse transcriptasepolymerase chain reaction (or RT-PCR, described in, for example, U.S.Pat. Nos. 5,322,770 and 5,310,652).

The PCR (or polymerase chain reaction) technique is well-known in theart and has been disclosed, for example, in Mullis and Faloona, MethodsEnzymol., 155:350-355 (1987). In its simplest form, PCR is an in vitromethod for the enzymatic synthesis of specific DNA sequences, using twoprimers that hybridize to opposite strands and flank the region ofinterest in the target DNA. A plurality of reaction cycles, each cyclecomprising: a denaturation step, an annealing step, and a polymerizationstep, results in the exponential accumulation of a specific DNAfragment, see for example, “PCR Protocols: A Guide to Methods andApplications”, Innis (Ed.), 1990, Academic Press: New York; “PCRStrategies”, Innis (Ed.), 1995, Academic Press: New York; “Polymerasechain reaction: basic principles and automation in PCR: A PracticalApproach”, McPherson et al. (Eds.), 1991, IRL Press: Oxford; Saiki etal., Nature 324:163-166 (1986). The termini of the amplified fragmentsare defined as the 5′ ends of the primers. Examples of DNA polymerasescapable of producing amplification products in PCR reactions include,but are not limited to: E. coli DNA polymerase I, Klenow fragment of DNApolymerase I, T4 DNA polymerase, thermostable DNA polymerases isolatedfrom Thermus aquaticus (Taq) which are available from a variety ofsources (for example, Perkin Elmer), Thermus thermophilus (United StatesBiochemicals), Bacillus stereothermophilus (Bio-Rad), or Thermococcuslitoralis (“Vent” polymerase, New England Biolabs). RNA target sequencesmay be amplified by reverse transcribing the mRNA into cDNA, and thenperforming PCR (RT-PCR), as described above. Alternatively, a singleenzyme may be used for both steps as described in U.S. Pat. No.5,322,770.

The duration and temperature of each step of a PCR cycle, as well as thenumber of cycles, are generally adjusted according to the stringencyrequirements in effect. Annealing temperature and timing are determinedboth by the efficiency with which a primer is expected to anneal to atemplate and the degree of mismatch that is to be tolerated. The abilityto optimize the reaction cycle conditions is well within the knowledgeof one of ordinary skill in the art. Although the number of reactioncycles may vary depending on the detection analysis being performed, itusually is at least 15, more usually at least 20, and may be as high as60 or higher. However, in many situations, the number of reaction cyclestypically ranges from about 20 to about 40.

The denaturation step of a PCR cycle generally comprises heating thereaction mixture to an elevated temperature and maintaining the mixtureat the elevated temperature for a period of time sufficient for anydouble-stranded or hybridized nucleic acid present in the reactionmixture to dissociate. For denaturation, the temperature of the reactionmixture is usually raised to, and maintained at, a temperature rangingfrom about 85° C. to about 100° C., usually from about 90° C. to about98° C., and more usually about 90° C. to about 94° C. for a period oftime ranging from about 3 to about 120 seconds, usually from about 5 toabout 30 seconds. In some embodiments, the first cycle is preceded by anelongated denaturation step ranging from about 1 to 10 minutes, usuallyfrom about 2 to 5 minutes.

Following denaturation, the reaction mixture is subjected to conditionssufficient for primer annealing to template DNA present in the mixture.The temperature to which the reaction mixture is lowered to achievethese conditions is usually chosen to provide optimal efficiency andspecificity, and generally ranges from about 45° C. to about 75° C.,usually from about 50° C. to about 70° C., and more usually from about53° C. to about 55° C. Annealing conditions are generally maintained fora period of time ranging from about 15 seconds to about 30 minutes,usually from about 30 seconds to about 1 minute.

Following annealing of primer to template DNA or during annealing ofprimer to template DNA, the reaction mixture is subjected to conditionssufficient to provide for polymerization of nucleotides to the primer'send in a such manner that the primer is extended in a 5′ to 3′ directionusing the DNA to which it is hybridized as a template (i.e., conditionssufficient for enzymatic production of primer extension product). Toachieve primer extension conditions, the temperature of the reactionmixture is typically raised to a temperature ranging from about 65° C.to about 75° C., usually from about 67° C. to about 73° C., andmaintained at that temperature for a period of time ranging from about15 seconds to about 20 minutes, usually from about 30 seconds to about 5minutes. In some embodiments, the final extension step is followed by anelongated extension step ranging from ranging from about 1 to 10minutes, usually from about 2 to 5 minutes.

The above cycles of denaturation, annealing, and polymerization may beperformed using an automated device typically known as a thermal cycleror thermocycler. Thermal cyclers that may be employed are described inU.S. Pat. Nos. 5,612,473; 5,602,756; 5,538,871; and 5,475,610. Thermalcyclers are commercially available, for example, from PerkinElmer-Applied Biosystems (Norwalk, Conn.), BioRad (Hercules, Calif.),Roche Applied Science (Indianapolis, Ind.), and Stratagene (La Jolla,Calif.).

In some embodiments, one or both of the PCR reactions are “kinetic PCR”(kPCR) or “kinetic RT-PCR” (kRT-PCR), which are also referred to as“real-time PCR” and “real-time RT-PCR,” respectively. These methodsinvolve detecting PCR products via a probe that provides a signal(typically a fluorescent signal) that is related to the amount ofamplified product in the sample. Examples of commonly used probes usedin kPCR and kRT-PCR include the following probes: TAQMAN® probes,Molecular Beacons probes, SCORPION® probes, and SYBR® Green probes.Briefly, TAQMAN® probes, Molecular Beacons, and SCORPION® probes eachhave a fluorescent reporter dye (also called a “fluor”) attached to the5′ end of the probes and a quencher moiety coupled to the 3′ end of theprobes. In the unhybridized state, the proximity of the fluor and thequench molecules prevents the detection of fluorescent signal from theprobe. During PCR, when the polymerase replicates a template on which aprobe is bound, the 5′-nuclease activity of the polymerase cleaves theprobe thus, increasing fluorescence with each replication cycle. SYBR®Green probes binds double-stranded DNA and upon excitation emit light;thus as PCR product accumulates, fluorescence increases.

In some embodiments, the PCR reaction is used in a “single-plex” PCRassay. “Single-plex” refers to a single assay that is not carried outsimultaneously with any other assays. Single-plex assays includeindividual assays that are carried out sequentially.

In some embodiments, the PCR reaction is used in a “multiplex” PCRassay. The term “multiplex” refers to multiple assays that are carriedout simultaneously, in which detection and analysis steps are generallyperformed in parallel. Within the context of the present disclosure, amultiplex assay will include the use of the primers, alone or incombination with additional primers to identify, for example, an HCVvirus variant along with one or more additional HCV variants or otherviruses.

In some embodiments, a first amplification step amplifies a region of atarget gene. In some embodiments the amplification product is less thanabout 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600, 500, 400,300, 250, 225, 200, 175 or 150 nucleotides long.

Nested PCR

In some embodiments, oligonucleotides of SEQ ID NOS: 1-36 and 39-45 canbe used in a “nested” PCR reaction to accurately amplify the NS3 gene ofHCV. A “nested” PCR reaction refers to a two-step specific amplificationof a target nucleic acid. In the first amplification step, a segment ofnucleic acid is amplified using a first (outer) primer set. In thesecond amplification step, a second (inner) primer set is used tofurther amplify a segment of the segment that was amplified in the firststep. Both first- and second-step primer sets will flank the targetnucleic acid. As a result, the final amplified product is obtainedwithin the frame of the segment that was amplified in the first step.The present disclosure may be used in conjunction with any nested PCRsystem known to those of skill in the art to generate an amplifiedtarget nucleic acid sequence. In some embodiments, the methods canemploy a reverse transcription step to produce cDNA, a firstamplification step performed with a first primer set which is specificto a target sequence, a second amplification step performed with asecond primer set on all or a portion of the first amplificationmixture, and optionally a sequencing step to determine the sequence ofthe target sequence.

In some embodiments, forward primers for a first amplification step ofnested PCR of the HCV NS3 gene are set forth in SEQ ID NOS: 21-25 and 43or any active fragments or combinations thereof. In some embodiments,reverse primers for a first amplification step of nested PCR of the HCVNS3 gene are set forth in SEQ ID NOS: 26-32 and 44-45 or any activefragments or combinations thereof.

In some embodiments, forward primers for a first amplification step ofnested PCR of the 1a genotype of the HCV NS3 gene are set forth in SEQID NOS: 1-5 and 39 or any active fragments or combinations thereof. Insome embodiments, reverse primers for a first amplification step ofnested PCR of the 1a genotype of the HCV NS3 gene are set forth in SEQID NOS: 6-10 and 40 or any active fragments or combinations thereof.

In some embodiments, forward primers for a first amplification step ofnested PCR of the 1b genotype of the HCV NS3 gene are set forth in SEQID NOS: 11-15 and 41 or any active fragments or combinations thereof. Insome embodiments, reverse primers for a first amplification step ofnested PCR of the 1a genotype of the HCV NS3 gene are set forth in SEQID NOS: 16-20 and 42 or any active fragments or combinations thereof.

In some embodiments, the forward and reverse primers comprise an outerprimer set for the first amplification step of HCV NS3. In someembodiments, this outer primer set comprises Primer sets 3-8, 13, or 14.

In some embodiments, the forward and reverse primers comprise an outerprimer set for the first amplification step of the 1a genotype of HCVNS3. In some embodiments, this outer primer set comprises Primer set 1or 11.

In some embodiments, the forward and reverse primers comprise an outerprimer set for the first amplification step of 1b genotype of HCV NS3.In some embodiments, this outer primer set comprises Primer set 2 or 12.

A second round of PCR amplification, e.g., in order to ensure PCRspecificity for the target sequence of interest, can be performed on theamplification product of the first amplification step. For instance, theamplicon, e.g., the HCV-specific amplicon, can be amplified in a PCRreaction with an inner primer set.

In some embodiments, forward primers for a second amplification step ofnested PCR of the 1a genotype of the HCV NS3 gene are set forth in SEQID NOS: 1-5, 33, and 39 or any active fragments or combinations thereof.In some embodiments, reverse primers for a second amplification step ofnested PCR of the 1a genotype of the HCV NS3 gene are set forth in SEQID NOS: 6-10, 34, and 40 or any active fragments or combinationsthereof.

In some embodiments, forward primers for a second amplification step ofnested PCR of the 1b genotype of the HCV NS3 gene are set forth in SEQID NOS: 11-15, 35, and 41 or any active fragments or combinationsthereof. In some embodiments, reverse primers for a second amplificationstep of nested PCR of the 1a genotype of the HCV NS3 gene are set forthin SEQ ID NOS: 16-20, 36, and 42 or any active fragments or combinationsthereof.

In some embodiments, the forward and reverse primers comprise an innerprimer set for the second amplification step of the 1a genotype of HCVNS3. In some embodiments, this inner primer set comprises Primer sets 1,9, or 11.

In some embodiments, the forward and reverse primers comprise an innerprimer set for the second amplification step of the 1b genotype of HCVNS3. In some embodiments, this inner primer set comprises Primer sets 2,10, or 12.

In some embodiments, the inner primer set is used to amplify anamplification product of the outer primer set. In some embodiments, theprimers of the inner primer set are homologous to the amplificationproduct of the outer primer set. In some embodiments, the inner primerset are homologous to the amplification product of the outer primer setand overlap with the outer primer set. In some embodiments, the innerprimer set are homologous to the amplification product of the outerprimer set and do not overlap with the outer primer set. In someembodiments, the outer primer set comprises Primer sets 3, 4, 5, 6, 7,8, 13, or 14 and the inner primer set comprises Primer sets 1, 2, 11, or12.

In some embodiments, the outer primer set comprises Primer set 1 or 11and the inner primer set comprises Primer set 9. In some embodiments,the outer primer set comprises Primer set 2 or 12 and the inner primerset comprises Primer set 10.

Detection of Amplification Products

Amplification products generated using the oligonucleotides and methodsof the present disclosure may be detected using a variety of methodsknown in the art.

In some embodiments, amplification products may simply be detected usingagarose gel electrophoresis and visualization by ethidium bromidestaining and exposure to ultraviolet (UV) light.

In some embodiments, the presence of a specific genotype can be shown byrestriction enzyme analysis. For example, a specific nucleotidepolymorphism can result in a nucleotide sequence comprising arestriction site which is absent from the nucleotide sequence of anotherallelic variant. Additionally or alternately, a specific nucleotidepolymorphism can result in the elimination of a nucleotide sequencecomprising a restriction site which is present in the nucleotidesequence of another allelic variant.

Examples of techniques for detecting differences of at least onenucleotide between two nucleic acids include, but are not limited to,selective oligonucleotide hybridization, selective amplification, orselective primer extension. For example, oligonucleotide probes may beprepared in which the known polymorphic nucleotide is placed centrallyand then hybridized to target DNA under conditions which permithybridization only if a perfect match is found, e.g., see Saiki et al.,Nature 324:163 (1986); Saiki et al., Proc. Natl Acad. Sci USA 86:6230(1989); and Wallace et al., Nucl. Acids Res. 6:3543 (1979). Suchspecific oligonucleotide hybridization techniques may be used for thesimultaneous detection of several nucleotide changes in differentpolymorphic regions of DNA. For example, oligonucleotides havingnucleotide sequences of specific allelic variants are attached to ahybridizing membrane and this membrane is then hybridized with labeledsample nucleic acid. Analysis of the hybridization signal will thenreveal the identity of the nucleotides of the sample nucleic acid.Alternatively unlabeled sample nucleic acid may be immobilized andcontacted with labeled oligonucleotides that hybridize selectively withspecific allelic variants.

Real-time pyrophosphate DNA sequencing is yet another approach todetection of polymorphisms and polymorphic variants, e.g., see Alderbornet al., Genome Research, 10(8):1249-1258 (2000). Additional methodsinclude, for example, PCR amplification in combination with denaturinghigh performance liquid chromatography (dHPLC), e.g., see Underhill etal., Genome Research, 7(10):996-1005 (1997).

In some embodiments, any of a variety of sequencing reactions known inthe art can be used to directly sequence at least a portion of amplifiedDNA and detect allelic variants. The sequence can be compared with thesequences of known allelic variants to determine which one(s) arepresent in the sample. Exemplary sequencing reactions include thosebased on techniques developed by Maxam and Gilbert, Proc. Natl. Acad.Sci USA, 74:560 (1977) or Sanger, Proc. Nat. Acad. Sci 74:5463 (1977).It is also contemplated that any of a variety of automated sequencingprocedures may be utilized when performing the subject assays, e.g., seeVenter et al., Science, 291:1304-1351 (2001); Lander et al., Nature,409:860-921 (2001), including sequencing by mass spectrometry, e.g., seeU.S. Pat. No. 5,547,835 and PCT Patent Publication No. WO 94/16101 andWO 94/21822; U.S. Pat. No. 5,605,798 and PCT Patent Application No.PCT/US96/03651; Cohen et al., Adv. Chromatogr. 36:127-162 (1996); andGriffin et al., Appl. Biochem. Biotechnol. 38:147-159 (1993). It will beevident to one skilled in the art that, for some embodiments, theoccurrence of only one, two or three of the nucleic acid bases need bedetermined in the sequencing reaction. Yet other sequencing methods aredisclosed, e.g., in U.S. Pat. Nos. 5,580,732; 5,571,676; 4,863,849;5,302,509; PCT Patent Application Nos. WO 91/06678 and WO 93/21340;Canard et al., Gene 148:1-6 (1994); Metzker et al., Nucleic AcidsResearch 22:4259-4267 (1994) and U.S. Pat. Nos. 5,740,341 and 6,306,597.

III—Kits

In some embodiments, the present disclosure provides kits comprisingmaterials useful for the amplification and detection or sequencing ofHCV NS3 according to methods described herein. The inventive kits may beused by diagnostic laboratories, experimental laboratories, orpractitioners.

Materials and reagents useful for the detection or sequencing of HCV NS3according to the present disclosure may be assembled together in a kit.In some embodiments, an inventive kit comprises at least one inventiveprimer set, and optionally, reverse transcription and/or amplificationreaction reagents. In some embodiments, a kit comprises reagents whichrender the procedure specific. Thus, a kit intended to be used for thedetection of a particular HCV variant (e.g., 1a or 1b) preferablycomprises primer sets described herein that can be used to amplify aparticular HCV target sequence of interest. A kit intended to be usedfor the multiplex detection of a plurality of HCV target sequencesand/or other viruses preferably comprises a plurality of primer sets(optionally in separate containers) described herein that can be used toamplify HCV target sequences described herein.

Suitable reverse transcription/amplification reaction reagents that canbe included in an inventive kit include, for example, one or more of:buffers; enzymes having reverse transcriptase and/or polymeraseactivity; enzyme cofactors such as magnesium or manganese; salts;nicotinamide adenide dinuclease (NAD); and deoxynucleoside triphosphates(dNTPs) such as, for example, deoxyadenosine triphospate; deoxyguanosinetriphosphate, deoxycytidine triphosphate and deoxythymidinetriphosphate, biotinylated dNTPs, suitable for carrying out theamplification reactions.

Depending on the procedure, the kit may further comprise one or more of:wash buffers and/or reagents, hybridization buffers and/or reagents,labeling buffers and/or reagents, and detection means. The buffersand/or reagents included in a kit are preferably optimized for theparticular amplification/detection technique for which the kit isintended. Protocols for using these buffers and reagents for performingdifferent steps of the procedure may also be included in the kit.

Furthermore, the kits may be provided with an internal control as acheck on the amplification procedure and to prevent occurrence of falsenegative test results due to failures in the amplification procedure. Anoptimal control sequence is selected in such a way that it will notcompete with the target nucleic acid sequence in the amplificationreaction (as described above).

Kits may also contain reagents for the isolation of nucleic acids frombiological specimen prior to amplification and/or for the purificationor separation of HCV before nucleic acid extraction.

The reagents may be supplied in a solid (e.g., lyophilized) or liquidform. The kits of the present disclosure optionally comprise differentcontainers (e.g., vial, ampoule, test tube, flask or bottle) for eachindividual buffer and/or reagent. Each component will generally besuitable as aliquoted in its respective container or provided in aconcentrated form. Other containers suitable for conducting certainsteps of the amplification/detection assay may also be provided. Theindividual containers of the kit are preferably maintained in closeconfinement for commercial sale.

The kit may also comprise instructions for using the amplificationreaction reagents and primer sets or primer/probe sets according to thepresent disclosure. Instructions for using the kit according to one ormore methods of the present disclosure may comprise instructions forprocessing the biological sample, extracting nucleic acid molecules,and/or performing the test; instructions for interpreting the results aswell as a notice in the form prescribed by a governmental agency (e.g.,FDA) regulating the manufacture, use or sale of pharmaceuticals orbiological products.

EXAMPLES Example 1 Nested PCR on NS3 Gene of HCV 1a

An assay was carried out to perform nested PCR on the NS3 gene of the 1avariant of HCV.

Viral RNA was extracted from 8 clinical samples using the QIAmp ViralRNA Kit (Qiagen). 140 μl of sample was used.

Reverse transcription (RT) PCR was then performed isolated HCV virus.37.8 μl nuclease free water, 23.6 μl dNTPs, 15.8 μl DTT, 4.7 μl of a 30μM equimolar mixture of SEQ ID NOS: 1-5, 4.7 μl of a 30 μM equimolarmixture of SEQ ID NOS: 6-10, and 7.9 μl of RNAse Inhibitor (AppliedBiosciences) were combined to create a first master mix. 6 μl of thefirst master mix was added to 20 μl of the viral template for eachclinical sample and the reaction was heated in the thermocycler for 2minutes at 90° C. and then 5 minutes at 52° C.

14 μl of a second master mix containing 157.5 μl RT-PCR Buffer, 7.9 μlRNAse Inhibitor (Applied Biosciences), 15.8 μl Superscript III ReverseTranscriptase (Invitrogen) and 39.4 μl DNA polymerase, was then added tothe reaction, resulting in a final concentration of 1,000 or 5,000 U/mlof the viral template. The RT reaction was completed by an additional 35minute incubation at 52° C. followed by 2 minutes at 94° C.

The PCR reaction then commenced immediately with 37 cycles of a 30second denaturation step at 94° C. followed by a 30 second annealingstep at 53° C. followed by a 2 minute extension step at 68° C. The lastcycle was followed by an additional 2 minute extension step at 68° C.and hold step at 4° C.

The RT-PCR product was run on an agarose gel to visualize the product.15 μl E-gel load dye was mixed with 5 μl of the amplification productand run on a gel for 30 minutes followed by visualization with UV light(see FIG. 1).

The nested PCR reaction was performed on the amplification products fromthe RT-PCR reaction. 1590 nuclease free water, 300 μl 10×PCR buffer, 60μl 10 mM dNTPs, 120 μl 25 mM MgCl₂, 300 μl 1 μM SEQ ID NO: 33, 300 μl 1μM SEQ ID NO: 34, and 30 μl 5U/μl AmpliTaq Gold (Applied Biosystems)were combined to create a master mix. 45 μl of master mix was added to 5μl of a 1:10 dilution of the RT-PCR amplification product.

The PCR reaction consisted of an initial 5 minute denaturation at 94° C.followed by 35 cycles of a 30 second denaturation step at 94° C.followed by a 30 second annealing step at 55° C. followed by a 2 minuteextension step at 72° C. The last cycle was followed by an additional 2minute extension step at 72° C. and hold step at 4° C.

Nested PCR products were examined by gel electrophoresis and subject tosequence analysis. FIG. 2 presents agarose gel electrophoresis profilesof samples taken after the second step of nested PCR.

Example 2 Nested PCR on NS3 Gene of HCV 1b

An assay was carried out to perform nested PCR on the NS3 gene of the 1bvariant of HCV.

Viral RNA was extracted from 6 clinical samples using the QIAmp ViralRNA Kit (Qiagen). 140 μl of sample was used.

Reverse Transcription (RT) PCR was then performed isolated HCV virus.57.6 μl nuclease free water, 36 μl dNTPs, 24 μl DTT, 7.2 μl of a 30 μMequimolar mixture of SEQ ID NOS: 11-15, 7.2 μl of a 30 μM equimolarmixture of SEQ ID NOS: 16-20, and 12 μl of RNAse Inhibitor (AppliedBiosciences) were combined to create a first master mix. 6 μl of thefirst master mix was added to 20 μl of the viral template for eachclinical sample and the reaction was heated in the thermocycler for 2minutes at 90° C. and then 5 minutes at 52° C.

14 μl of a second master mix containing 240 μl RT-PCR Buffer, 12 μlRNAse Inhibitor (Applied Biosciences), 24 μl Superscript III ReverseTranscriptase (Invitrogen) and 60 μl DNA polymerase, was then added tothe reaction, resulting in a final concentration of 10,000 or 5,000 U/mlof the viral template. The RT reaction was completed by an additional 35minute incubation at 52° C. followed by 2 minutes at 94° C.

The PCR reaction then commenced immediately with 37 cycles of a 30second denaturation step at 94° C. followed by a 30 second annealingstep at 53° C. followed by a 2 minute extension step at 68° C. The lastcycle was followed by an additional 2 minute extension step at 68° C.and hold step at 4° C.

The RT-PCR product was run on an agarose gel to visualize the product.15 μl E-gel load dye was mixed with 5 μl of the amplification productand run on a gel for 30 minutes followed by visualization with UV light(see FIG. 3).

The nested PCR reaction was performed on the amplification products fromthe RT-PCR reaction. 1590 nuclease free water, 300 μl 10× PCR buffer, 60μl 10 mM dNTPs, 120 μl 25 mM MgCl2, 300 μl 1 μM SEQ ID NO: 35, 300 μl 1μM SEQ ID NO: 36, and 30 μl 5U/μl AmpliTaq Gold (Applied Biosystems)were combined to create a master mix. 45 μl of master mix was added to 5μl of a 1:10 dilution of the RT-PCR amplification product.

The PCR reaction consisted of an initial 5 minute denaturation at 94° C.followed by 35 cycles of a 30 second denaturation step at 94° C.followed by a 30 second annealing step at 52° C. followed by a 2 minuteextension step at 72° C. The last cycle was followed by an additional 2minute extension step at 72° C. and hold step at 4° C.

Nested PCR products were examined by gel electrophoresis and subject tosequence analysis. FIG. 4 presents agarose gel electrophoresis profilesof samples taken after the second step of nested PCR.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the inventions described herein. The scope of the presentdisclosure is not intended to be limited to the scope of the abovedescription, but rather is as set forth in the following claims.

What is claimed is:
 1. A collection of primers for amplifying a portionof an HCV NS3 1a genomic sequence comprising: one or more forwardprimers each comprising a nucleic acid sequence selected from the groupconsisting of: (i) SEQ ID NO: 1, (ii) SEQ ID NO: 2 or an active fragmentthereof, (iii) SEQ ID NO: 3 or an active fragment thereof, and (iv) SEQID NO: 4; and one or more reverse primers each comprising a nucleic acidsequence selected from the group consisting of: (i) SEQ ID NO: 6 or anactive fragment thereof, (ii) SEQ ID NO: 7 or an active fragmentthereof, (iii) SEQ ID NO: 8 or an active fragment thereof, and (iv) SEQID NO: 9; wherein the one or more forward primers and the one or morereverse primers are deoxyribonucleic acid primers, and wherein an activefragment is 15 or more nucleotides in length.
 2. A collection of primersfor amplifying a portion of an HCV NS3 1a genomic sequence, wherein thecollection of primers comprises forward primers and reverse primers, theforward primers comprising: a primer comprising SEQ ID NO: 1 or anactive fragment thereof, a primer comprising SEQ ID NO: 2 or an activefragment thereof, a primer comprising SEQ ID NO: 3 or an active fragmentthereof, a primer comprising SEQ ID NO: 4 or an active fragment thereof,and a primer comprising SEQ ID NO: 5 or an active fragment thereof, andthe reverse primers comprising a primer comprising SEQ ID NO: 6 or anactive fragment thereof, a primer comprising SEQ ID NO: 7 or an activefragment thereof, a primer comprising SEQ ID NO: 8 or an active fragmentthereof, a primer comprising SEQ ID NO: 9 or an active fragment thereof,and a primer comprising SEQ ID NO: 10 or an active fragment thereof. 3.A kit for amplifying HCV NS3 by nested PCR comprising a set of innerprimers and a set of outer primers, wherein the set of outer primerscomprises a collection of primers as defined in claim
 1. 4. A kit foramplifying HCV NS3 by nested PCR comprising a set of inner primers and aset of outer primers, wherein the set of outer primers comprises acollection of primers as defined in claim
 2. 5. The kit of claim 3,wherein at least some of the inner primers are labeled with a detectablemoiety.
 6. The kit of claim 3, wherein at least some of the innerprimers are tagged with tag sequences.
 7. The kit of claim 6, whereinthe tag sequences are M13 tag sequences.
 8. A method of amplifying HCVNS3 in a sample comprising contacting a sample with a set of outerprimers, wherein the set of outer primers comprises the collection ofprimers of claim 1 and submitting the resulting mixture to a firstnucleic acid amplification reaction.
 9. The method of claim 8, furthercomprising contacting a product from the first nucleic acidamplification reaction with a set of inner primers and submitting theresulting mixture to a second nucleic acid amplification reaction. 10.The method of claim 8, wherein the set of outer primers comprises acollection of primers as defined in claim
 2. 11. The method of claim 9,wherein at least some of the inner primers are labeled.
 12. The methodof claim 9, wherein at least some of the inner primers are tagged withtag sequences.
 13. The method of claim 12, wherein the tag sequences areM13 tag sequences.
 14. The method of claim 9, further comprisingsequencing a product from the second nucleic acid amplification reactionto detect an HCV NS3 polymorphism.